Alpha beta T cell recognition of Major Histocompatibility Complex molecules presenting peptide antigens plays an important role in many aspects of adaptive cellular immunity, and is vital to health in mammals. While a great deal has been learned about the structural biology of alpha beta T cell receptors in the last ten years, there remain many unresolved issues concerning how these molecules distinguish between the many peptide-MHC complexes that they need to survey, and how the pMHC recognition event is structurally conveyed into a signal. In this renewal application we wish to continue our efforts to understand the interplay of alpha beta TCR structure, recognition and signaling by taking advantage of a variety of traditional (X-ray crystallography) and novel (combinatorial biology, NMR, in situ affinity measurements) approaches to this problem. We are focusing on two cornerstones of T cell-mediated immunity: 1- the inherent reactivity of the alpha beta TCR repertoire for MHC presenting diverse peptide antigens (Aims #1, #2 and #3), and 2- the nature of the subsequent communication of alpha beta TCR/MHC ligation to the associated CD3 subunits (Aim #4). In prior terms of this award we gained new insight into the structural basis of alloreactivity, MHC bias, TCR cross-reactivity, and autoimmunity. Collectively, our recent studies suggest a paradigm-changing hypothesis: that TCR reactivity with MHC may be attributable to specific, germline-encoded recognition rules between residues on the TCR Variable region CDR1 and 2 loops, and residues on the MHC helices. That TCR reactivity with MHC may be describable by [unreadable]structural rules[unreadable] could greatly simplify our understanding of this complex process. In the current term, we challenge this hypothesis by diversifying the scope of structural and mechanistic studies to ask how several different TCR germline-derived V-region interactions with MHC are influenced by diversity of chain pairing, CDR3 and peptide sequences. We also investigate the dynamics of TCR/MHC scanning using NMR. As a means of identifying novel collections of peptides reactive with a given TCR to explore the issue of TCR cross-reactivity, we have developed a yeast combinatorial biology approach that has many practical applications, including peptide identification for [unreadable]orphan[unreadable] TCRs. Finally, in order to begin to bridge recognition with signaling, we take direct aim, using a novel affinity-maturation approach, at obtaining high-resolution structural information for how the alpha beta TCR ectodomains associate with the CD3 ectodomains within the context of the transmembrane complex.